Enhanced functionality and delivery of a protein from a porous substrate

ABSTRACT

Compositions, methods, articles of manufacture, and kits are provided for storage and delivery of proteins.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/858,488, filed Jul. 25, 2013, the contents of which are herebyincorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

During storage, a biomolecule should resist aggregation, denaturation,and degradation in order to remain stable. Biomolecules exhibit highlyvariable stability depending on the relative purity of the biomolecules,the identity of any contaminating biomolecules, the primary, secondary,and tertiary structure of the biomolecules, and the environment in whichthey are stored.

Methods of storage include precipitation and/or crystallization of thebiomolecule. For example, biomolecules (e.g., proteins) may beprecipitated and/or crystallized by a precipitant such as ammoniumsulfate and stored for later reconstitution. Alternatively, biomoleculescan be lypholized and stored in a solid form. As yet anotheralternative, biomolecules can be stored in solution, such as in abuffered solution. Biomolecules that have been stored in a suitablemanner, such that the biomolecule (or a substantial fraction thereof) isstable, can then be delivered from the storage medium, or extracted, andsubsequently utilized for a variety of purposes.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method ofdelivering a protein, the method comprising: providing a poroussubstrate comprising a reversibly immobilized protein and a proteinaggregation modifying agent; and eluting the reversibly immobilizedprotein from the porous substrate with an eluent, thereby delivering theprotein.

In some cases, the eluent comprises a buffered solution at a pH abovethe isoelectric point of the reversibly immobilized protein. In somecases, the eluent comprises a protein aggregation modifying agentselected from the group consisting of a cyclodextrin, a non-ionicsurfactant, an ionic surfactant, a zwitterionic surfactant, anon-detergent sulfobetaine, a simple sugar, a polysaccharide, a polyol,an organic solvent, an aggregation modifying protein, a disorderedpeptide sequence, an amino acid, an oxido-reduction agent, alyoprotectant, a cryoprotectant, and a chaotropic agent. In some cases,the eluent comprises a western blot transfer buffer. The western blottransfer buffer can contain tris(hydroxymethyl)aminomethane (Tris),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, methanol,ethanol, or propanol. The western blot transfer buffer can contain twoor more compounds selected from the group consisting oftris-(hydroxymethyl)-aminomethane (Tris),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, methanol,ethanol, and propanol.

In some cases, the eluting comprises subjecting the substrate todiffusion, capillary action, vacuum, pressure, or electrophoresis, orcontacting the porous substrate with a magnetic particle that has anaffinity for the reversibly immobilized protein. In some cases, theeluting comprises eluting a portion of the protein onto a membrane. Insome cases, the method comprises eluting at least 50% of the reversiblyimmobilized protein from the porous substrate with an eluent. In somecases, the method comprises eluting substantially all of the reversiblyimmobilized protein from the porous substrate with an eluent. In somecases, the eluted reversibly immobilized protein is less aggregated incomparison to a protein eluted from a porous substrate that does notcomprise a protein aggregation modifying agent.

In some cases, the membrane has an immobilized protein on its surface.In some cases, the non-specific binding sites on the membrane areblocked. In some cases, the eluting comprises contacting the substratewith the membrane and transferring the protein reversibly immobilized onthe substrate to the membrane via diffusion, capillary action, vacuum,pressure, or the application of an electromagnetic force. In some cases,the method further comprises washing non-specifically bound and unboundeluted protein from the membrane. In some cases, the method furthercomprises detecting the presence of the eluted protein on the membrane.

In some cases, the method further comprises binding the protein elutedfrom the substrate to the immobilized protein on the membrane surface;and detecting the presence of the eluted protein on the membrane,thereby detecting the presence of the immobilized protein on themembrane surface. In some cases, 1, 2, 3, 4 or more different proteinsare reversibly immobilized on the porous substrate. In some cases, 1, 2,3, 4 or more different proteins are reversibly immobilized on 1, 2, 3,4, or more regions, e.g., laterally delimited regions, of the poroussubstrate.

In some cases, the porous substrate comprises a composition selectedfrom the group consisting of whatman paper, paper, a cellulose filter, aglass microfiber filter, nitrocellulose, polyvinylidene difluoride, asintered glass, a sintered polymer, a sintered metal, a spunboundpolyester, rayon, nylon, a porous polymer monolith, a porous polymerbead, a capillary wicking bed, natural or synthetic sponge, andfiberglass. In some cases, the capillary wicking bed is a thin-filmchromatography plate.

In some cases, the porous substrate comprises a mask region and areversible immobilization region, wherein reversibly immobilized proteinis immobilized on the reversible immobilization region. In some cases,the reversibly immobilized protein is reversibly immobilized across atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, all, or substantiallyall of the reversible immobilization region. In some cases, the poroussubstrate further comprises 2, 3, 4 or more reversible immobilizationregions. In some cases, the porous substrate further comprises a borderregion. In some cases, the mask region comprises a hydrophobic polymer.In some cases, the mask region comprises a fatty acid or wax. In somecases, the reversibly immobilized protein is evenly distributed acrossat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, substantially all,or all of at least one surface of the substrate.

In some cases, the reversibly immobilized protein is reversiblyimmobilized on the porous substrate for less than about 12 hours, 24hours, 48 hours, or less than about 1 week. In some cases, the proteinis reversibly immobilized on the porous substrate for at least about 1week, 1 month, 3 months, 6 months, 9 months, or 1 year.

In some cases, the reversibly immobilized protein is an antibody. Insome cases, the antibody is reversibly immobilized on the poroussubstrate and no other antibody is immobilized on the porous substrate.In some cases, less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%,substantially no, or no protein is irreversibly immobilized on theporous substrate. In some cases, at least 50%, 60%, 70%, 80%, 90%, 95%,99%, substantially all, or all of the reversibly immobilized protein isnon-aggregated and non-denatured. In some cases, the reversiblyimmobilized protein is unlabeled. In some cases, the reversiblyimmobilized protein is labeled.

In some cases, the protein aggregation modifying agent is selected fromthe group consisting of a cyclodextrin, a non-ionic surfactant, an ionicsurfactant, a zwitterionic surfactant, a non-detergent sulfobetaine, asimple sugar, a polysaccharide, a polyol, an organic solvent, anaggregation modifying protein, a disordered peptide sequence, an aminoacid, an oxido-reduction agent, a lyoprotectant, a cryoprotectant, and achaotropic agent.

The cyclodextrin can be selected from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, andsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin.

The ionic surfactant can be sodium dodecyl sulfate or sodium octylsulfate. The non-ionic surfactant can be selected from the groupconsisting of polysorbate 80, polysorbate 20, Brij-35, and apolyoxypropylene-polyoxyethylene block co-polymer. The non-detergentsulfobetaine can be selected from the group consisting of NDSB 256, NDSB221, NDSB 211, NDSB 201, NDSB 195,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate, 3-(Benzyldimethylammonio)propanesulfonate, and Dimethylethylammoniumpropane sulfonate. The simplesugar can be selected from the group consisting of sucrose, mannitol,sorbitol, inositol, xylitol, erythritol, glucose, galactose, raffinose,and trehalose. The polysaccharide can be selected from the groupconsisting of dextran, starch, and hydroxyethyl starch. The polyol canbe selected from the group consisting of glycerol, ethylene glycol,polyethylene glycol, pentaerythritol propoxylate, and pentaerythritolpropoxylate. The organic solvent can be selected from the groupconsisting of ethanol, butanol, propanol, dimethyl formamide,2-methyl-2,4-pentanediol, 2,3-butanediol, 1,2-propanediol,1,6-hexanediol, and dimethyl sulfoxide. The aggregation modifyingprotein can be selected from the group consisting of albumin, casein,gelatin, ubiquitin, lysozyme, and a late embryogenesis abundant (LEA)protein. The amino acid can be selected from the group consisting ofglycine, proline, taurine, arginine, cystine, and cysteine. Theoxido-reduction agent can be selected from the group consisting ofmercaptoethanol, dithiothreitol, dithioerythriotl,tris(2-carboxyethyl)phosphine, glutathione, glutathione disulfide, andCu²⁺. And the lyoprotectant, cryoprotectant, or chaotropic agent can beselected from the group consisting of urea, thiourea, guanidinium,calcium, bromide, iodide, chloride, potassium, thiocyanate, perchlorate,chlorate, trimethylamine N-oxide, and phenol.

In some embodiments, the present invention provides a method of storinga protein the method comprising: providing a porous substrate and aprotein aggregation modifying agent; contacting the porous substrate inthe presence of the protein aggregation modifying agent with a solutioncontaining a protein; and reversibly immobilizing the protein on theporous substrate, thereby storing the reversibly immobilized protein onthe porous substrate.

In some cases, at least 50% of the reversibly immobilized protein issubstantially non-denatured and substantially non-aggregated. In somecases, 1, 2, 3, or 4 or more different proteins are reversiblyimmobilized on the porous substrate. In some cases, the porous substratecomprises a composition selected from the group consisting of whatmanpaper, paper, a cellulose filter, a glass microfiber filter,nitrocellulose, polyvinylidene difluoride, a sintered glass, a sinteredpolymer, a sintered metal, a spunbound polyester, rayon, nylon, a porouspolymer monolith, a porous polymer bead, a capillary wicking bed,natural or synthetic sponge, and fiberglass. In some cases, thecapillary wicking bed is a thin-film chromatography plate.

In some cases, the porous substrate comprises a mask region, and areversible immobilization region, wherein the method comprisesreversibly immobilizing the protein on the reversible immobilizationregion. In some cases, the reversibly immobilized protein is reversiblyimmobilized across at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,or substantially all of the reversible immobilization region. In somecases, the mask region comprises a hydrophobic polymer. In some cases,the mask region comprises a fatty acid or wax. In some cases, the poroussubstrate further comprises a border region. In some cases, thereversibly immobilized protein is evenly distributed across at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or all of at least onesurface of the substrate.

In some cases, the reversible immobilization comprises drying the poroussubstrate after contacting the porous substrate in the presence of theprotein aggregation modifying agent with the solution containing theprotein. In some cases, the drying comprises air drying, vacuum drying,drying in an oven at 30, 40, 50, 60, or 65° C., or lypholization. Insome cases, the protein aggregation modifying agent substantiallyprotects the reversibly immobilized protein from aggregation anddenaturation during the drying. In some cases, the reversiblyimmobilized protein is stored on the porous substrate for at least about1 week, 1 month, or 6 months. In some cases, the reversibly immobilizedprotein is stored on the porous substrate for less than about 12 hours,24 hours, 48 hours, or 1 week.

In some cases, the protein is an antibody. In some cases, the antibodyis reversibly immobilized on the porous substrate and no other antibodyis immobilized on the porous substrate. In some cases, 2, 3, or 4 ormore antibodies are reversibly immobilized on the porous substrate. Insome cases, substantially no protein is irreversibly immobilized on theporous substrate. In some cases, the protein is unlabeled. In somecases, the protein is labeled.

In some cases, the porous substrate is configured to elute thereversibly immobilized protein when contacted with an eluent. In somecases, the contacting the porous substrate in the presence of theprotein aggregation modifying agent with a solution containing a proteincomprises contacting the solution containing the protein with a poroussubstrate that contains a protein aggregation modifying agent. In somecases, the contacting the porous substrate in the presence of theprotein aggregation modifying agent with a solution containing a proteincomprises contacting the porous substrate with a solution containing aprotein and a protein aggregation modifying agent.

In some cases, the protein aggregation modifying agent is selected fromthe group consisting of a cyclodextrin, a non-ionic surfactant, an ionicsurfactant, a zwitterionic surfactant, a non-detergent sulfobetaine, asimple sugar, a polysaccharide, a polyol, an organic solvent, anaggregation modifying protein, a disordered peptide sequence, an aminoacid, an oxido-reduction agent, a lyoprotectant, a cryoprotectant, and achaotropic agent.

The cyclodextrin can be selected from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, andsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin.

The ionic surfactant can be sodium dodecyl sulfate or sodium octylsulfate. The non-ionic surfactant can be selected from the groupconsisting of polysorbate 80, polysorbate 20, Brij-35, and apolyoxypropylene-polyoxyethylene block co-polymer. The non-detergentsulfobetaine can be selected from the group consisting of NDSB 256, NDSB221, NDSB 211, NDSB 201, NDSB 195,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate, 3-(Benzyldimethylammonio)propanesulfonate, and Dimethylethylammoniumpropane sulfonate. The simplesugar can be selected from the group consisting of sucrose, mannitol,sorbitol, inositol, xylitol, erythritol, glucose, galactose, raffinose,and trehalose. The polysaccharide can be selected from the groupconsisting of dextran, starch, and hydroxyethyl starch. The polyol canbe selected from the group consisting of glycerol, ethylene glycol,polyethylene glycol, pentaerythritol propoxylate, and pentaerythritolpropoxylate. The organic solvent can be selected from the groupconsisting of ethanol, butanol, propanol, dimethyl formamide,2-methyl-2,4-pentanediol, 2,3-butanediol, 1,2-propanediol,1,6-hexanediol, and dimethyl sulfoxide. The aggregation modifyingprotein can be selected from the group consisting of albumin, casein,gelatin, ubiquitin, lysozyme, and a late embryogenesis abundant (LEA)protein. The amino acid can be selected from the group consisting ofglycine, proline, taurine, arginine, cystine, and cysteine. Theoxido-reduction agent can be selected from the group consisting ofmercaptoethanol, dithiothreitol, dithioerythritol,tris(2-carboxyethyl)phosphine, glutathione, glutathione disulfide, andCu²⁺. The lyoprotectant, cryoprotectant, or chaotropic agent can beselected from the group consisting of urea, thiourea, guanidinium,calcium, bromide, iodide, chloride, potassium, thiocyanate, perchlorate,chlorate, trimethylamine N-oxide, and phenol.

In some embodiments, the present invention provides an article ofmanufacture comprising a porous substrate, a protein reversiblyimmobilized on a surface of the substrate, and a protein aggregationmodifier, wherein the article is made by any of the foregoing methods.In some cases, the protein reversibly immobilized on the surface of thesubstrate is dry. In some cases, the substrate is at least about 5-15 cmwide and at least about 5-15 cm long.

In some embodiments, the present invention provides a kit comprising anyof the foregoing articles of manufacture and an elution buffer. In somecases, the elution buffer comprises a protein aggregation modifierselected from the group consisting of a cyclodextrin, a non-ionicsurfactant, an ionic surfactant, a zwitterionic surfactant, anon-detergent sulfobetaine, a simple sugar, a polysaccharide, a polyol,an organic solvent, an aggregation modifying protein, a disorderedpeptide sequence, an amino acid, an oxido-reduction agent, alyoprotectant, a cryoprotectant, and a chaotropic agent. In some cases,the elution buffer comprises a western blot transfer buffer.

In some embodiments, the present invention provides a kit comprising apackage of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of any of theforegoing articles of manufacture.

In some embodiments, the present invention provides a system fordelivering protein comprising: any of the foregoing articles ofmanufacture; a membrane having a second protein immobilized on asurface; a transfer buffer; and an apparatus adapted to transfer thereversibly immobilized protein on the substrate to the membrane viadiffusion, capillary action, vacuum, pressure, or electromagnetic force.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: depicts the use of the protein aggregation modifying agentcyclodextrin to inhibit the formation of antibody aggregates formed bywetting a porous substrate with a solution containing the antibody. Asillustrated, this results in an increased delivery of non-aggregatedantibody and more complete removal of antibody from the substrate.

FIG. 2: depicts delivery of antibody from a porous substrate to a gel ora membrane. As illustrated by gray lines, the gel or the membrane cancontain immobilized target proteins.

FIG. 3: demonstrates the effect of methyl-beta-cyclodextrin (MBCD) onthe transfer efficiency of antibody out of Whatman® chromatographypaper.

FIG. 4: depicts one method of spatially controlled antibody delivery toa membrane.

FIG. 5: depicts the use of wax barriers to deliver multiple antibodiesto a membrane in a spatially controlled manner.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “reversibly immobilized” as used herein refers to a biomolecule(e.g., a protein) that has been immobilized on a substrate (e.g., aporous substrate) such that it can be eluted from the substrate. Thereversibly immobilized biomolecule can be immobilized such that it canbe eluted without loss of activity (e.g., binding or enzymaticactivity), or without substantial loss of activity (e.g., retains atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, all, or substantiallyall of its binding or enzymatic activity prior to reversibleimmobilization). For example, the reversibly immobilized biomolecule canbe immobilized such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, all, or substantially all of the biomolecule can be elutedfrom the substrate in a non-denatured or non-aggregated form.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The term “western blot” includes not only the standard western blot, butalso variants such as far-western blot, Northwestern blot andSouthwestern blot. In general, a western blot involves the transfer of aprotein to a membrane, and subsequent detection of the protein on themembrane. There are a variety of membranes suitable for use as westernblot membranes known in the art including, without limitation,polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane,polyamide membrane, polyester membrane, and nylon membrane. Westernblotting typically utilizes a transfer buffer. There are a variety ofwestern blot transfer buffers known in the art. In general, the westernblot transfer buffer will have a pH that is above the isoelectric pointof the protein to be transferred. Thus, when a voltage potential isapplied, the protein will migrate toward the positive electrode.Alternatively, the transfer buffer can have a pH below the isoelectricpoint of the protien to be transferred. In such cases, the protein willmigrate toward the negative electrode.

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

The term “antibody” as used herein also includes antibody fragments thatretain binding specificity. For example, there are a number of wellcharacterized antibody fragments. Thus, for example, pepsin digests anantibody C-terminal to the disulfide linkages in the hinge region toproduce F(ab′)2, a dimer of Fab which itself is a light chain joined toVH-CH1 by a disulfide bond. The F(ab′)2 may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo by utilizing recombinant DNA methodology or chemically. Thus,the term “antibody”, as used here includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized usingrecombinant DNA methodologies.

Antibodies as used here also include various V_(H)-V_(L) pair formats,including single chain antibodies (antibodies that exist as a singlepolypeptide chain), e.g., single chain Fv antibodies (sFv or scFv), inwhich a variable heavy and a variable light region are joined together(directly or through a peptide linker) to form a continuous polypeptide.The single chain Fv antibody is a covalently linked V_(H)-V_(L) that maybe expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker(e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988).While the V_(H) and V_(L) are connected to each as a single polypeptidechain, the V_(H) and V_(L) domains associate non-covalently. An antibodycan also be in another fragment form, such as a disulfide-stabilized Fv(dsFv). Other fragments can also be generated, e.g., using recombinanttechniques, as soluble proteins or as fragments obtained from displaymethods. Antibodies can also include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such asantibodies from camelids or sharks. Since the V_(H) region of a heavychain dimer IgG in a camelid or shark does not have to make hydrophobicinteractions with a light chain, the region in the heavy chain thatnormally contacts a light chain is changed to hydrophilic amino acidresidues. V_(H) domains of heavy-chain dimer IgGs are called VHH(camelid) or VNAR (shark) domains. Antibodies for use in the currentinvention additionally include single domain antibodies (dAbs) andnanobodies (see, e.g., Cortez-Retamozo, et al., Cancer Res.64:2853-2857, 2004).

II. Introduction

Proteins can be stored and delivered in a variety of ways. For example,proteins may be stored in a solution, as a lyophilized solid, orimmobilized onto a substrate. In any case, it is desirable to employstorage and delivery conditions that protect the stability of theprotein. For example, proteins are generally stored in a manner thatreduces protein denaturation and aggregation. Protein denaturation oraggregation can be induced by a variety of phenomenon including heat,cold, ionic strength, pH, the presence of a denaturant, and contact witha hydrophobic interface. One of the most hydrophobic interfaces is theair water interface. Consequently, it is generally desirable to reduceprotein aggregation during storage and delivery by minimizing contactwith air-water interfaces.

During protein storage and delivery, a protein can encounter an airwater interface at many points. For example, protein aggregation can beinduced by contact with an air water interface during mixing orpipetting of a protein. Similarly, protein aggregation can be induced bycontact with an air water interface simply while residing in a containeras a protein solution. In particular, storage mechanisms in which aprotein is contacted with a solid surface substrate can introduce anextremely large air-water interface. For example, a protein in solutionthat is contacted with a substrate will be subject to an increasinglylarge air water interface to volume ratio as the volume of the liquid inwhich the protein is solubilized decreases during the drying step.

Similarly, a protein dried onto a substrate can experience aggregationduring a subsequent rewetting step, such as prior to, or during,delivery of the stored protein. For example, as the substrate iscontacted with a wetting solution (e.g., an eluent), there will exist alarge air-water interface. As explained above, interaction between theprotein and the air-water interface can lead to protein denaturationand/or aggregation. Porous substrates in particular exacerbate thisair-water interface problem because the interstitial spaces of thesubstrate lead to a large air-water interface.

The inventors have discovered that aggregation of a protein on asubstrate (e.g., a porous substrate) can be surprisingly reduced oreliminated by the presence of protein aggregation modifying agents.Without wishing to be bound by theory, it is believed that proteinaggregation modifying agents that act to displace proteins from theair-water interface, and thereby protect them from denaturation andaggregation, are particularly effective in reducing the aggregation ofproteins immobilized on a porous substrate.

Accordingly, described herein are methods and compositions for storingproteins on and/or within a porous substrate and delivering suchproteins under conditions that reduce or eliminate aggregation ordenaturation. Such conditions include the use of one or more proteinaggregation modifying agents as described herein. Also provided hereinare compositions containing a protein stored on a porous substrate suchthat the protein is protected from aggregation or denaturation by thepresence of a protein aggregation modifying agent. Also described hereinare kits containing multiple compositions containing a protein stored ona porous substrate. Such compositions and kits can, e.g., be used todeliver a protein to a membrane, a container, or other assay platformfor subsequent analysis. The specification also describes kits forstoring proteins on a porous substrate in the presence of a proteinaggregation modifying agent.

III. Compositions

Described herein are compositions containing a porous substrate, aprotein, and a protein aggregation modifying agent for storage ordelivery of the protein.

A. Proteins

The protein to be stored or delivered can be any protein. For example,the protein can be an antibody, an affimer, a lipocalin (e.g., ananticalin), thioredoxin A, bilin binding protein, or a proteincontaining an ankyrin repeat, the Z domain of staphylococcal protein A,or a fibronectin type III domain. The protein can also be an enzyme, areporter protein, a receptor, a hormone, a toxin, a cytokine, or adetection reagent. The protein can be labeled or unlabeled as describedherein. The protein can be purified from a biological source orsynthesized. For example, the protein can be produced in a cell, e.g.,heterologously produced in a cell and purified therefrom. In some cases,the protein is a recombinant protein.

In some embodiments, the protein can be a polypeptide containing atleast 10-15 amino acids. Such a polypeptide can be purified from abiological source or produced by synthetic methods. Alternatively, theprotein can be a longer polypeptide, e.g., at least 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, or 100 amino acids. For example, the polypeptidecan be between 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 80-90, or90-100 amino acids in length. In some embodiments, the protein can be atleast about 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,1250, 1500, 2000, 2500, 3000, 5000 amino acids in length or more.

In some embodiments, the protein can be a protein fragment. For example,the protein can consist of a one or more domains of a larger protein. Insome cases, the protein can consist of a fragment that contains thedesired activity. For example, the protein can consist of a fragmentthat contains a binding or enzymatic activity.

Labels

The proteins described herein can be detected by detecting a label thatis linked to the protein. The label can be linked directly to theprotein (e.g., by a covalent bond) or the attachment can be indirect(e.g., using a chelator or linker molecule). The terms “label” and“detectable label” are used synonymously herein. In some embodiments,each protein label (e.g., a first label linked to a first protein, asecond label linked to a second protein, etc.) generates a detectablesignal and the signals (e.g., a first signal generated by the firstlabel, a second signal generated by the second label, etc.) aredistinguishable. In some embodiments, the two or more protein labelscomprise the same type of agent (e.g., a first label that is a firstfluorescent agent and a second label that is a second fluorescentagent). In some embodiments, the two or more protein labels (e.g., thefirst label, second label, etc.) combine to produce a detectable signalthat is not generated in the absence of one or more of the labels.

Examples of detectable labels include, but are not limited to,biotin/streptavidin labels, nucleic acid (e.g., oligonucleotide) labels,chemically reactive labels, fluorescent labels, enzyme labels,radioactive labels, quantum dots, polymer dots, mass labels, andcombinations thereof. In some embodiments, the label can include anoptical agent such as a chromophore, fluorescent agent, phosphorescentagent, chemiluminescent agent, etc. Numerous agents (e.g., dyes, probes,or indicators) are known in the art and can be used in the presentinvention. (See, e.g., Invitrogen, The Handbook—A Guide to FluorescentProbes and Labeling Technologies, Tenth Edition (2005)). Chromophoresinclude co-enzymes or co-factors that have a detectable absorbance. Insome cases, a protein can be detected by detecting the intrinsicabsorbance of a peptide bond at, e.g., 220 or 280 nm.

Fluorescent agents can include a variety of organic and/or inorganicsmall molecules or a variety of fluorescent proteins and derivativesthereof. For example, fluorescent agents can include but are not limitedto cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines,phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines,fluoresceins (e.g., FITC, 5-carboxyfluorescein, and6-carboxyfluorescein), benzoporphyrins, squaraines, dipyrrolopyrimidones, tetracenes, quinolines, pyrazines, corrins, croconiums,acridones, phenanthridines, rhodamines (e.g., TAMRA, TMR, and RhodamineRed), acridines, anthraquinones, chalcogenopyrylium analogues, chlorins,naphthalocyanines, methine dyes, indolenium dyes, azo compounds,azulenes, azaazulenes, triphenyl methane dyes, indoles, benzoindoles,indocarbocyanines, benzoindocarbocyanines, BODIPY™ and BODIPY™derivatives, and analogs thereof. In some embodiments, a fluorescentagent is an Alexa Fluor dye. In some embodiments, a fluorescent agent isa polymer dot or a quantum dot. Fluorescent dyes and fluorescent labelreagents include those which are commercially available, e.g., fromInvitrogen/Molecular Probes (Eugene, Oreg.) and Pierce Biotechnology,Inc. (Rockford, Ill.). In some embodiments, the optical agent is anintercalating dye. In some embodiments, 2, 3, 4, 5, or more proteins areeach labeled with an optical agent such as a fluorescent agent (e.g., afirst protein labeled with a first fluorescent label, a second proteinlabeled with a second fluorescent label, etc.), and each protein that islabeled with an optical agent is detected by detecting a signalgenerated by the optical agent (e.g., a fluorescent signal generated bya fluorescent label). In some embodiments, all of the proteins arelabeled with an optical agent, and each optical agent-labeled protein isdetected by detecting a signal generated by the optical agent.

In some embodiments, the label is a radioisotope. Radioisotopes includeradionuclides that emit gamma rays, positrons, beta and alpha particles,and X-rays. Suitable radionuclides include but are not limited to ²²⁵Ac,⁷²As, ²¹¹At, ¹¹B, ¹²⁸Ba, ²¹²Bi, ⁷⁵Br, ⁷⁷Br, ¹⁴C, ¹⁰⁹Cd, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ¹⁸F, ⁶⁷Ga, ⁶⁸Ga, ³H, ¹⁶⁶Ho, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹¹¹In,¹⁷⁷Lu, ¹³N, ¹⁵O, ³²P, ³³P, ²¹²Pb, ¹⁰³Pd, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm,⁸⁹Sr, ^(99m)Tc, ⁸⁸Y and ⁹⁰Y. In some embodiments, 2, 3, 4, 5, or moreproteins are each labeled with a radioisotope (e.g., a first proteinlabeled with a first radioisotope, a second protein labeled with asecond radioisotope, etc.), and each protein that is labeled with aradioisotope is detected by detecting radioactivity generated by theradioisotope. For example, one protein can be labeled with a gammaemitter and one protein can be labeled with a beta emitter.Alternatively, the proteins can be labeled with radioinuclides that emitthe same particle (e.g., alpha, beta, or gamma) at different energies,where the different energies are distinguishable. In some embodiments,all of the proteins are labeled with a radioisotope and each labeledprotein can be detected by detecting radioactivity generated by theradioisotope.

In some embodiments, the label is an enzyme, and the protein is detectedby detecting a product generated by the enzyme. Examples of suitableenzymes include, but are not limited to, urease, alkaline phosphatase,(horseradish) hydrogen peroxidase (HRP), glucose oxidase,β-galactosidase, luciferase, alkaline phosphatase, and an esterase thathydrolyzes fluorescein diacetate. For example, a horseradish-peroxidasedetection system can be used with the chromogenic substratetetramethylbenzidine (TMB), which yields a soluble product in thepresence of hydrogen peroxide that is detectable at 450 nm. An alkalinephosphatase detection system can be used with the chromogenic substratep-nitrophenyl phosphate, which yields a soluble product readilydetectable at 405 nm. A β-galactosidase detection system can be usedwith the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside(ONPG), which yields a soluble product detectable at 410 nm. A ureasedetection system can be used with a substrate such as urea-bromocresolpurple (Sigma Immunochemicals; St. Louis, Mo.). In some embodiments, 2,3, 4, 5, or more proteins are each labeled with an enzyme (e.g., a firstprotein labeled with a first enzyme, a second protein labeled with asecond enzyme, etc.), and each protein that is labeled with an enzyme isdetected by detecting a product generated by the enzyme. In someembodiments, all of the proteins are labeled with an enzyme, and eachenzyme-labeled protein is detected by detecting a product generated bythe enzyme.

In some embodiments, the label is an affinity tag. Examples of suitableaffinity tags include, but are not limited to, biotin, peptide tags(e.g., FLAG-tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Strep-tag,eXact-tag), and protein tags (e.g., GST-tag, MBP-tag, GFP-tag).

In some embodiments, the label is a nucleic acid label. Examples ofsuitable nucleic acid labels include, but are not limited to,oligonucleotide sequences, single-stranded DNA, double-stranded DNA, RNA(e.g., mRNA or miRNA), or DNA-RNA hybrids. In some embodiments, thenucleic acid label is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,or 1000 nucleotides in length.

In some embodiments, the label is a nucleic acid barcode. As used hereina “barcode” is a short nucleotide sequence (e.g., at least about 4, 6,8, 10, or 12, nucleotides long) that uniquely defines a labeledmolecule, or a second molecule bound to the labeled protein. The lengthof the barcode sequence determines how many unique samples can bedifferentiated. For example, a 4 nucleotide barcode can differentiate 44or 256 samples or less, a 6 nucleotide barcode can differentiate 4096different samples or less, and an 8 nucleotide barcode can index 65,536different samples or less. The use of barcode technology is well knownin the art, see for example Katsuyuki Shiroguchi, et al. Digital RNAsequencing minimizes sequence-dependent bias and amplification noisewith optimized single-molecule barcodes, PNAS (2012); and Smith, A M etal. Highly-multiplexed barcode sequencing: an efficient method forparallel analysis of pooled samples, Nucleic Acids Research Can 11,(2010).

In some embodiments, the label is a “click” chemistry moiety. Clickchemistry uses simple, robust reactions, such as the copper-catalyzedcycloaddition of azides and alkynes, to create intermolecular linkages.For a review of click chemistry, see Kolb et al., Agnew Chem40:2004-2021 (2001). In some embodiments, a click chemistry moiety(e.g., an azide or alkyne moiety) can be detected using anotherdetectable label (e.g., a fluorescently labeled, biotinylated, orradiolabeled alkyne or azide moiety).

Techniques for attaching detectable labels to proteins are well known.For example, a review of common protein labeling techniques can be foundin Biochemical Techniques: Theory and Practice, John F. Robyt andBernard J. White, Waveland Press, Inc. (1987). Other labeling techniquesare reviewed in, e.g., R. Haugland, Excited States of Biopolymers,Steiner ed., Plenum Press (1983); Fluorogenic Probe Design andSynthesis: A Technical Guide, PE Applied Biosystems (1996); and G. T.Herman, Bioconjugate Techniques, Academic Press (1996).

In some embodiments, two or more protein labels (e.g., a first label,second label, etc.) combine to produce a detectable signal that is notgenerated in the absence of one or more of the labels. For example, insome embodiments, each of the labels is an enzyme, and the activities ofthe enzymes combine to generate a detectable signal that is indicativeof the presence of the labels (and thus, is indicative of each of thelabeled proteins). Examples of enzymes combining to generate adetectable signal include coupled assays, such as a coupled assay usinghexokinase and glucose-6-phosphate dehydrogenase; and a chemiluminescentassay for NAD(P)H coupled to a glucose-6-phosphate dehydrogenase,beta-D-galactosidase, or alkaline phosphatase assay. See, e.g., Maeda etal., J Biolumin Chemilumin 1989, 4:140-148.

B. Substrates

As described herein, a protein can be stored on, or delivered from, asubstrate under conditions that reduce protein aggregation ordenaturation. The protein can be stored on, or delivered from, anysuitable substrate. In general, the substrate must be mechanically andchemically stable enough to provide a platform for protein storage anddelivery. Also, the substrate compositions described herein generallyprovide for reversible immobilization of the protein such that at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, all, or substantially allof the protein can be eluted from the substrate. In some cases, thesubstrate composition is selected so that all, substantially all, or atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the proteinremains free, substantially free, or at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% free of aggregates.

The substrate can be any size and shape. For example, the substrate canbe circular, oval, square, or rectangular. In some cases, the substrateis a rectangular strip, e.g., a chromatography strip. For example, thesubstrate can be a strip of about 0.25, 0.5, or 1 cm in width. In somecases, the strip can be used to deliver a protein to a region, a lane,or a number of lanes of a gel (e.g., a polyacrylamide gel) or a membrane(e.g., a nitrocellulose, PVDF, or nylon membrane). In general, thesubstrate will have three substantial dimensions (e.g., a measurablelength, width, and thickness, or a measurable radius and thickness). Insome cases, the substrate is roughly planar in that it has twodimensions (e.g., length and width) that are each at least about 2-fold,5-fold, 10-fold, 100-fold, or more larger than the third dimension(e.g., thickness). In some cases, the substrate can be sized for use ina blotting apparatus, such as a Southern or western blotting apparatus.In some cases, the substrate is a membrane. Alternatively, the substratecan consist of one or more beads.

In some embodiments, the substrate is sized to provide for ease ofmanipulation by hand. In other embodiments, the substrate can be smallerthan that which is readily manipulated by hand. For example, thesubstrate can be either larger than about 1 cm×1 cm in length and width(e.g., larger than about 2 cm in any dimension, or larger than about 5cm in any dimension) and thus easy to handle. Alternatively, thesubstrate can be smaller than about 1 cm×1 cm (e.g., smaller than about0.5 cm×0.5 cm or smaller than about 0.1 cm×0.1 cm) in length and widthand thus suitable for micromanipulation techniques.

Exemplary sizes include substrates that are at least about 0.25 cm, 0.5cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 10 cm, 12 cm, 15 cm,20 cm, 30 cm or more in at least one dimension. Exemplary sizes furtherinclude substrates that are about 1 cm×1 cm, 7×8.4 cm, 8.5×13.5 cm, 10cm×15 cm, or 25×28 cm in length and width respectively. Exemplary sizesfurther include 8.5 cm×9 cm, 7 cm×9 cm, 8 cm×10.7 cm, 10 cm×10 cm, 7cm×8.5 cm, 8.3 cm×7.3 cm, 8 cm×8 cm, 8.3 cm×13 cm, 10.8 cm×13.5 cm. Insome cases, the substrate is configured to deliver protein to amicrotiter plate (e.g., a 6, 8, 12, 24, 48, 96, 384, or 1536 wellplate). For example, the substrate may be sized to the dimensions ofsuch a microtiter plate. In some cases, the substrate is configured todeliver protein to a microscope slide, or a microarray. For example, thesubstrate may be sized to the dimensions of such a microtiter plate.

The substrate can be a porous substrate. Porous substrates generallyhave a large surface area due to the presence of a plurality of pores.The large surface area can increase the protein loading capacity of thesubstrate. In the absence of a protein aggregation modifier, however,the large surface area of the porous substrate can also increase proteinaggregation due to the increased air-water interface.

In some embodiments, the porous substrate has a large surface area ascompared to a nonporous substrate of the same material and size. Forexample, the porous substrate can have at least about a 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 500, 1000-fold ormore increased surface area as compared to a nonporous substrate of thesame material and size. In some embodiments, the porous substrate has alarge surface specific surface area. For example, the porous substratecan have a specific surface area of at least about 0.1 m²/g, 0.5 m²/g, 1m²/g, 10 m²/g, or more as measured by standard techniques.

In some embodiments, the porous substrate possesses a high specificbinding capacity for protein. For example, in some cases, the poroussubstrate can immobilize at least about 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10mg, 15 mg, 20 mg, 40 mg, 60 mg, 100 mg, or more protein for every mg ofsubstrate material.

In some embodiments, the porous substrate can have a particular poresize, a particular average pore size, or a particular pore size range.For example, the porous substrate can contain 0.1 μm pores, 0.2 μmpores, 0.45 μm pores, or 1, 2, 4, 5, 6, 7, 8, 10, 15, 20 μm pores, orpores larger than about 20 μm. As another example, the porous substratecan contain pores that average 0.1, 0.2, 0.45, 1, 2, 4, 5, 6, 7, 8, 10,15, or 20 μm, or more in size. As another example, the porous substratecan contain pores that range about 0.1-8 μm, 0.2-8 μm, 0.45-8 μm, 1-8μm, 0.1-4 μm, 0.1-2 μm, 0.1-1 μm, 0.1-0.45 μm, 0.2-8 μm, 0.2-4 μm, 0.2-2μm, 0.2-1 μm, 0.2-0.45 μm, 0.45-8 μm, 0.45-4 μm, 0.45-2 μm, 0.45-1 μm insize. In some cases, the porous substrate can contain pores that areless than about 20 μm in size. For example, the porous substrate canconsist of a material in which at least about 50%, 60%, 70%, 80%, 90% ormore of the pores are less than about 20, 15, 10, or 5 μm in size. Insome cases, the pores are large enough to contain one or more proteinsof average size (e.g., about 1 nm). For example, the pores can be atleast 1 nm in size, at least 5 nm in size, at least 10, 100, or 500 nmin size. Alternatively, at least 50%, 60%, 70%, 80%, 90% or more of thepores can be more than 1, 5, 10, 50, 100, or 500 nm in size. As usedherein, pore size can be measured as a radius or a diameter.

The substrate can be treated or functionalized to provide higher proteinloading, directed protein loading, or specificity for a particularprotein. For example, the substrate, or a portion thereof, can betreated to alter the hydrophilicity or alter the hydrophobicity of thetreated area. In some cases, altering the hydrophilicity orhydrophobicity of a substrate can increase protein loading, create maskregions in which protein is not loaded, direct flow of proteins when thesubstrate is wet, or reduce protein denaturation or aggregation.

In addition to mask regions in which the protein is not reversiblyimmobilized, the substrate can also contain a border region. In somecases, the border region is marked or annotated to provide orientationinformation or describe the type or nature of the reversibly immobilizedprotein. In some cases, the border region can be configured to provide aregion for handling the substrate. For example, the border region canserve as a useful handle for manual or robotic manipulation of thesubstrate.

Mask regions and border regions, e.g., laterally delimited regions, aredescribed, for example, in U.S. application Ser. No. 13/950,590 filed onJul. 25, 2013, herein incorporated by reference in its entirety.

The substrate can be marked or annotated such that the origin,composition, or location of the reversibly immobilized protein isrecorded. For example, one or more mask regions can be visuallydiscernible, such that one of skill in the art can determine thelocation of the reversibly immobilized protein. Alternatively, theprotein name, identity, amount, lot number, etc. can be printed orstamped on a portion of the substrate. In some cases, the substrate ismarked or annotated such that the proper orientation for transfer, e.g.,in a transfer apparatus, of the reversibly immobilized protein to asecond substrate or a membrane is discernible.

As another example, the substrate, or a portion thereof, can befunctionalized with a ligand to which a protein will bind or that willbind to a protein. Exemplary ligands include, but are not limited to,ligands which interact with antibodies, e.g., protein A, G, or L.Exemplary ligands also include polypeptide antigen tags, e.g., a myc,HA, FLAG, S, SBP, V5, Softag, Avitag, calmodulin, His, Xpress, TC, or Tytag. One of skill in the art will recognize that a wide variety ofligands can be used herein, and the choice of a particular ligand willdepend on the protein to be stored or delivered.

As yet another example, the substrate, or a portion thereof, can befunctionalized to provide an ion exchange utility. For example, thesubstrate, or a portion thereof, can be functionalized to increase thebinding of cations, anions, or cations and anions. Functional groupssuitable for use in providing an ion exchange utility include tertiaryand quaternary amines for soft and hard anion exchange respectively.Functional groups suitable for use in providing an ion exchange utilityalso include carboxylic acids and sulphonates for soft and hard cationexchange respectively.

Substrates include, but are not limited to, polymer containingsubstrates. The polymer can consist of polymer beads, a polymermembrane, or a polymer monolith. In some cases, the polymer iscellulose. Cellulose containing substrates include paper, cloth, woven,or non-woven cellulose substrates. Cloth substrates include thosecontaining a natural cellulose fiber such as cotton or wool. Papersubstrates include those containing natural cellulose fiber (e.g.,cellulose or regenerated cellulose) and those containing cellulose fiberderivatives including, but not limited to cellulose esters (e.g.,nitrocellulose, cellulose acetate, cellulose triacetate, celluloseproprionate, cellulose acetate propionate, cellulose acetate butyrate,and cellulose sulfate) and cellulose ethers (e.g., methylcellulose,ethylcellulose, ethyl methyl cellulose, hydroxyethyl cellulose,hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethylhydroxyethyl cellulose, and carboxymethyl cellulose). In some cases, thecellulose substrate contains rayon. In some cases, the substrate ispaper, such as a variety of WHATMAN® paper.

Substrates further include substrates that contain a sintered material.For example, the substrate can contain a sintered glass, a sinteredpolymer, or sintered metal. In some cases, the sintered material isformed by sintering one or more of powdered glass, powdered polymer, orpowdered metal. In other cases, the sintered material is formed bysintering one or more of glass, metal, or polymer fibers. In still othercases, the sintered material is formed from the sintering of one or moreof glass, polymer, or metal beads.

Substrates can also contain one or more non-cellulosic polymers, e.g. asynthetic polymer, a natural polymer, or a semisynthetic polymer. Forexample, the substrate can contain a polyester, such as polyglycolide,polylactic acid, polycaprolactone, polyethylene adipate,polyhydroxylalkanoate, polyhydroxybutyrate,poly(3-hydroxybutyrate-co-3-hydroxyvalerate, polyethylene terephthalate,polybutylene terephthalate, polytrimethylene terephthalate, polyethylenenaphthalate, Vectran®. In some cases, the polymer is spunbound, such asa spunbound polyester.

Additional synthetic polymers include, but are not limited to nylon,polypropylene, polyethylene, polystyrene, divinylbenzene, polyvinyl,polyvinyl difluoride, high density polyvinyl difluoride, polyacrylamide,a (C2-C6) monoolefin polymer, a vinylaromatic polymer, avinylaminoaromatic polymer, a vinylhalide polymer, a (C1-C6) alkyl(meth)acrylate polymer, a(meth)acrylamide polymer, a vinyl pyrrolidonepolymer, a vinyl pyridine polymer, a (C1-C6)hydroxyalkyl (meth)acrylatepolymer, a (meth)acrylic acid polymer, an acrylamidomethylpropylsulfonicacid polymer, an N-hydroxy-containing (C1-C6) alkyl(meth)acrylamidepolymer, acrylonitrile or a mixture of any of the foregoing.

Substrates can also contain one or more polysaccharides. Exemplarypolysaccharides include those containing cellulose, agarose, amylose,chitin, chitosan, galactosamine, curdlan, dextran, xylan, inulin, andderivatives thereof, e.g., esters, phenyl carbamates, alkyl carabmates,and benzyl carbamates. In some cases, the polysaccharides arecross-linked. For example, the substrate can include agarose, or a crosslinked agarose. In some cases, the substrate can include cross linksbetween the polysaccharide and other constituents of the substrate.

Substrates also include capillary wicking beds and materials usedtherein. For example, the substrate can include a thin layerchromatography plate, or be formed of any of the thin layerchromatography substrates known in the art. Thin layer chromatographysubstrates known in the art include, but are not limited to, silica,silica derivatized with C4, C8, or C18 alkyl groups, and alumina.

Substrates can also contain glass, glass fibers, fiberglass, natural orsynthetic sponge, silica, alumina, or a derivative thereof.

In addition to the foregoing substrate materials, the substrate can alsocontain any combination of the foregoing. In some cases, the substratecan contain composite materials that include a combination of materialsdescribed above. For example, the substrate can contain glass or silicafibers in a synthetic polymer matrix.

In some embodiments, the compositions described herein includesubstrates on which a protein is reversibly immobilized thereon. Forexample, the protein can be eluted from the substrate under appropriateconditions. In some cases, the compositions provided herein can providefor a reversibly immobilized protein that can be eluted from thesubstrate without the formation, without substantial formation, orwherein less than about 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1% of theeluted protein is aggregated.

In some embodiments, the substrate is not a lateral flow assaysubstrate. For example, in some cases, the substrate does not contain anantibody that is never eluted from the substrate during use. In somecases, the substrate does not contain an antibody that is irreversiblyimmobilized to the substrate or is not elutable from the substrate. Insome cases, the substrate does not host a binding reaction or enzymaticreaction.

C. Protein Aggregation Modifying Agents

As described herein, protein aggregation modifying agents can beutilized to reduce or eliminate aggregation or denaturation of proteinsstored on, or delivered from, a substrate. In some cases, proteinaggregation modifying agents that act to displace proteins from theair-water interface and thereby protect them from denaturation andaggregation are particularly effective in reducing the aggregation ofproteins immobilized on a porous substrate. In other cases, the proteinaggregation modifying agent directly affects the stability of theprotein by binding to the protein and/or stabilizing the protein. Inother cases, the protein aggregation modifying agent acts to shift theequilibrium away from the denatured or unfolded state and thus reduceaggregation. For example, in some cases, the interaction between theprotein aggregation modifying agent and the protein is thermodynamicallydisfavored due to strong repulsion between the amide backbone of theprotein and the protein aggregation modifying agent. Thus, unfolding ofthe protein in the presence of the protein aggregation modifying agentis disfavored because unfolding exposes more protein amide backbonesurface to the protein aggregation modifying agent.

Protein aggregation modifying agents can include one or more of acyclodextrin, a non-ionic surfactant, an ionic surfactant, azwitterionic surfactant, a non-detergent sulfobetaine, a simple sugar, apolysaccharide, a polyol, an organic solvent, an aggregation modifyingprotein, a disordered peptide sequence, an amino acid, anoxido-reduction agent, a lyoprotectant, a cryoprotectant, and achaotropic agent.

Cyclodextrins include, but are not limited to, α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, (2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, andsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin. Cyclodextrins can alsoinclude cyclodextrin polymers containing one or more of the foregoingcyclodextrin molecules. Additional cyclodextrins are known in the art,and include, e.g. those described on the world wide web atcyclodextrin.com. Exemplary concentrations of cyclodextrins include, butare not limited to about 1 mM, 2 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, 15 mM,20 mM, 25 mM, 50 mM, 75 mM, or 100 mM.

Non-ionic surfactants include polyethylen-sorbitan-fatty acid esters,polyethylene-polypropylene glycols and polyoxyethylene-stearates.Polyethylen-sorbitan-fatty acid esters includepolyethylen(20)-sorbitan-esters (Tween 20™) andpolyoxyethylene(20)-sorbitanmonooleate (Tween 80™).Polyethylene-polypropylene glycols includepolyoxypropylene-polyoxyethylene block co-polymers such as those soldunder the names Pluronic® or Poloxamer™. Polyoxyethylene-stearatesinclude those sold under the trademark Myrj™. Polyoxyethylene monolaurylethers include those sold under the trademark Brij™, e.g., Brij-35.Exemplary concentrations of non-ionic surfactants include, but are notlimited to about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%,2.5%, 5%, 7.5%, and about 10% w/w, w/v, or v/v.

Ionic surfactants include anionic surfactants and cationic surfactants.Anionic surfactants useful in the present invention include, but are notlimited to soaps including alkali soaps, such as sodium, potassium andammonium salts of aliphatic carboxylic acids, usually fatty acids, suchas sodium stearate. Additional anionic surfactants include organic aminesoaps such as organic amine salts of aliphatic carboxylic acids, usuallyfatty acids, such as triethanolamine stearate. Cationic surfactantsuseful in the present invention include, but are not limited to, aminesalts such as octadecyl ammonium chloride and quarternary ammoniumcompounds such as benzalkonium chloride. Ionic surfactants furtherinclude the sodium, potassium and ammonium salts of alkyl sulfates, suchas sodium dodecyl sulfate and sodium octyl sulfate. Exemplaryconcentrations of ionic surfactants include, but are not limited toabout 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%,7.5%, and about 10% w/w, w/v, or v/v.

Zwitterionic surfactants have both cationic and anionic centers attachedto the same molecule. The cationic part is, e.g., based on primary,secondary, or tertiary amines or quaternary ammonium cations. Theanionic part can include sulfonates, as in CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate). Otheranionic groups are sultaines illustrated by cocamidopropylhydroxysultaine and betaines, e.g., cocamidoethyl betaine,cocamidopropyl betaine, or lauramidopropyl betaine. Exemplaryconcentrations of zwitterionic surfactants include, but are not limitedto about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%,7.5%, and about 10% w/w, w/v, or v/v.

Non detergent sulfobetaines (NDSBs) have a sulfobetaine hydrophilicgroup and a short hydrophobic group that cannot aggregate to formmicelles, therefore NDSBs are not considered detergents. NDSBs include,but are not limited to NDSB 256, NDSB 221, NDSB 211, NDSB 201, NDSB 195,3-(4-tert-Butyl-1-pyridinio)-1-propanesulfonate,3-(1-pyridinio)-1-propanesulfonate, 3-(Benzyldimethylammonio)propanesulfonate, and Dimethylethylammoniumpropane sulfonate. Exemplaryconcentrations of NDSBs include, but are not limited to about 0.01%,0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, and about10% w/w, w/v, or v/v.

Polyols are compounds with multiple hydroxyl functional groups. In somecases, polyols can modify the aggregation or denaturation behavior of aprotein by a variety of mechanisms. For example, in some cases, thepolyol can shift the equilibrium to the folded state by presenting athermodynamically disfavored interaction with the protein backbone.Alternatively, in some cases, the polyol can bind to and stabilize thefolded state of the protein.

Polyols can include simple sugars such as sucrose, mannitol, sorbitol,inositol, xylitol, erythritol, glucose, galactose, raffinose, andtrehalose. Polyols can also include polysaccharides such as dextran,starch, hydroxyethyl starch, and polymers containing the simple sugarsdescribed herein. Polyols can also include glycerol, ethylene glycol,polyethylene glycol, pentaerythritol propoxylate, and pentaerythritolpropoxylate, and combinations thereof.

Organic solvents include, but are not limited to, those organic solventthat are known to inhibit denaturation, unfolding, or aggregation of oneor more proteins. A variety of suitable organic solvents are known inthe art. For example, organic solvents can include ethanol, butanol,propanol, phenol, dimethyl formamide, 2-methyl-2,4-pentanediol,2,3-butanediol, 1,2-propanediol, 1,6-hexanediol, and dimethyl sulfoxide.

Aggregation modifying proteins include proteins known in the art toinhibit denaturation, unfolding, or aggregation of one or more proteins.Aggregation modifying proteins include, but are not limited to,albumins. Albumins are proteins that are water-soluble, are moderatelysoluble in concentrated salt solutions, and experience heatdenaturation. Albumins include serum albumins (e.g., bovine, horse, orhuman serum albumin) and egg albumin (e.g., hen egg-white albumin).Aggregation modifying proteins also include casein, gelatin, ubiquitin,lysozyme, and late embryogenesis abundant (LEA) proteins. LEA proteinsinclude LEA I, LEA II, LEA III, LEA IV, LEA V, and atypical LEAproteins. LEA proteins are known in the art and described, e.g., inGoyal K., et al., Biochemical Journal 288(pt. 1), 151-57, (2005).

Protein aggregation modifying agents include amino acids. In some cases,the amino acids can serve an oxido-reduction function to maintain anappropriate oxidative potential for the protein immobilized on thesubstrate. Suitable oxido-reducitve amino acids include cysteine andcystine. Other amino acids serve to reduce denaturation or aggregationthrough a non-oxido-reductive method. For example, arginine, glycine,proline, and taurine have been shown to reduce protein aggregation.

Other oxido-reduction agents can be employed to reduce proteinaggregation. Oxido-reductants other than cysteine and cystine, can beused to optimize the reduction potential in the substrate onto which theprotein is immobilized. Additional oxido-reductants includemercaptoethanol, dithiothreitol, dithioerythritol,tris(2-carboxyethyl)phosphine, glutathione, glutathione disulfide, andoxidized derivatives thereof, as well as Cu²⁺.

Protein aggregation modifying agents can also include lyoprotectants,cryoprotectants, or chaotropic agents. In some cases, the proteinaggregation modifying agent is a chaotrope such as urea, thiourea,guanidinium, cyanate, thiocyanate, trimethylammonium,tetramethylammonium, cesium, rubidium, nitrate, acetate, iodide,bromide, trichloroacetate, or perchlorate. Under certain conditions,such as at low concentrations, chaotropes can reduce proteinaggregation. Other protein aggregation modifying agents includetrimethylamine N-oxide.

Protein aggregation modifying agents can further include salts. Saltsinclude, but not limited to, the sodium, potassium, magnesium, andcalcium salts of chloride, sulfate, and phosphate. Protein aggregationmodifying agents can further include buffering agents. Exemplarybuffering agents include, but are not limited to, tris (hydroxymethyl)amino methene (TRIS), TAPSO, MES, HEPES, PIPES, CAPS, CAPSO, MOPS,MOPSO, and sodium or potassium phosphate, carbonate, bicarbonate,citrate, acetate, or borate buffers.

The protein aggregation modifying agents can be provided in any suitableconcentration. In some cases, the protein is provided as an aqueoussolution containing protein and protein aggregation modifying agents. Insuch cases, the solution can be contacted with a substrate and,optionally, dried. Exemplary concentrations of protein aggregationmodifying agents in the aqueous protein solution include, but are notlimited to, about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 4%,5%, 10%, 20%, or about 25% or more w/v of the solution. Furtherexemplary concentrations include, but are not limited to, about 1 μM, 5μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 300 μM, 500 μM,750 μM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM,500 mM, and 1M.

In some cases, the protein aggregation modifying agents are provided onthe substrate. Exemplary compositions containing a protein aggregationmodifying agent and a substrate include, substrates that contain about0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or about10%, 20%, or about 25% by weight of protein aggregation modifyingagents.

Protein aggregation modifying agents can be provided in any suitablecombination. For example, in some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more of the foregoing protein aggregation modifying agents can beutilized to reduce aggregation of a protein immobilized on a substrate.In some cases, prior to contacting the substrate with the proteinsolution, the substrate contains a protein aggregation modifying agent,and the protein solution contains a different protein aggregationmodifying agent. In other cases, the substrate contains a proteinaggregation modifying agent and the protein solution contain the sameprotein aggregation modifying agent. In some cases, prior to contactingthe substrate with the protein solution, the substrate contains aprotein aggregation modifying agent, and the protein solution does notcontain a protein aggregation modifying agent. In some cases, prior tocontacting the substrate with the protein solution, the protein solutioncontains a protein aggregation modifying agent and the substrate doesnot.

D. Eluent

In some embodiments, eluents (e.g., elution buffers) can be used forobtaining a protein that has been stored (e.g., reversibly immobilized)on a substrate. The eluents described herein are generally water orbuffered aqueous solutions that are capable of wetting the substrate andsolubilizing the reversibly immobilized proteins. Virtually any pHbuffering composition that is soluble in water and generally compatiblewith proteins can be utilized. Exemplary buffering agents include, butare not limited to, tris (hydroxymethyl) amino methene (TRIS), TAPSO,MES, HEPES, PIPES, CAPS, CAPSO, MOPS, MOPSO, and sodium or potassiumphosphate, carbonate, bicarbonate, citrate, acetate, or borate buffers.

The eluent can also contain various compositions that aid in proteinsolubilization or stabilization. For example, the eluent can contain anyof the foregoing protein aggregation modifying agents. Alternatively,the eluent (e.g., water) can acquire a protein solubilization orstabilization function by dissolving buffers and/or protein aggregationmodifying agents present on the substrate. In any case, theconcentration of one or more protein aggregation modifying agentspresent in an eluent can be the same or different from the concentrationutilized during the reversible immobilization process.

The eluent can be a transfer buffer for transferring the reversiblyimmobilized protein to a second substrate or to a membrane. One of skillin the art will understand that there are a wide variety of suitabletransfer buffer compositions, such as a western blot transfer buffer.Western blot transfer buffer commonly contains one or more oftris-(hydroxymethyl)-aminomethane (Tris),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, methanol,ethanol, and propanol. In some cases, western blot transfer buffer canalso contain a low concentration of denaturing agent such as a detergent(e.g., sodium dodecyl sulfate) or a chaotrope (e.g., urea). In caseswhere the transfer will occur in response to an applied voltagepotential, the transfer buffer can be buffered at a pH that is above theisoelectric point of the one or more reversibly immobilized proteins sothat the transfer occurs toward the positive electrode. Alternatively,the transfer buffer can be buffered at a pH that is below theisoelectric point of the one or more reversibly immobilized proteins sothat the transfer occurs toward the negative electrode.

IV. Methods

Described herein are methods of storing or delivering proteins that areimmobilized (e.g., reversibly immobilized) on a substrate. In general,these methods rely on the inventors' surprising discovery that thepresence of certain compositions, referred to herein as proteinaggregation modifying agents, reduce or eliminate aggregation of theprotein during the process of reversibly immobilizing the protein ontothe substrate, the process of storing the reversibly immobilized proteinon the substrate, the process of wetting a substrate containing aprotein, or the process of eluting the protein from the substrate.

A. Storage

In some embodiments, one or more proteins are stored on a substrate byreversibly immobilizing the protein onto the substrate. In someembodiments, the method includes:

providing a porous substrate and a protein aggregation modifying agent;contacting the porous substrate in the presence of the proteinaggregation modifying agent with a solution containing a protein; andreversibly immobilizing the protein on the porous substrate, therebystoring the reversibly immobilized protein on the porous substrate.

The method includes providing or obtaining a suitable substrate.Suitable substrates are described above and include various poroussubstrates. In some cases, the substrate is provided dry. In othercases, the substrate is provided wet. For example, the substrate can bewetted with a solution containing a protein aggregation modifying agent.In other cases, the substrate is wetted before contact with the proteinwith a solution containing a protein aggregation modifying agent and thedried before contact with the protein. In some cases, the providing isperformed by ordering or obtaining a substrate suitable for the storageor delivery from another party.

The method further includes contacting the porous substrate with asolution containing the protein. As explained above, the protein can bevirtually any protein or combination of proteins. For example, theprotein can be an antibody, or 2, 3, 4, 5, or more antibodies. In somecases, the substrate has only one antibody immobilized, only oneantibody reversibly immobilized, only two antibodies immobilized, oronly two antibodies reversibly immobilized on the substrate.

The contacting can be performed in any manner desired. Such contactingsteps can include spraying a protein solution onto one or two sides, ora portion thereof, of the substrate, dipping at least a portion of thesubstrate into the protein solution, brushing the protein solution ontoat least a portion of the substrate, coating at least a portion of thesubstrate with the protein solution, pouring or pipetting. In somecases, the spraying, dipping, brushing, coating, pouring, or pipettingis performed multiple times for each protein to be reversiblyimmobilized onto the substrate. In other cases, a protein solutioncontaining all the proteins to be reversibly immobilized onto thesubstrate is utilized. In still other cases, multiple contacting stepsare performed with proteins solutions of the same or differingcompositions.

In some cases, the contacting is performed manually. For example, thesubstrate can be dipped into a suitable protein solution manually, orthe solution can be pipetted or sprayed onto the substrate manually. Inother cases, the contacting is performed using an instrument, or anautomated or robotic platform. For example, a pipetting instrument, aspraying instrument (e.g., a modified ink-jet apparatus), or otherautomated or robotic platform.

In some cases, the contacting is performed such that one or moreproteins are reversibly immobilized in a spatially addressable fashion.For example, the one or more proteins may be sprayed from an ink-jetmodified to spray solutions containing a biomolecule, applied via aprecision pipetting instrument, or the contacting can be performedmanually such that the protein is reversibly immobilized at a specifiedlocation, which location can be determined or tracked after thereversible immobilization. In some cases, the substrate can be contactedin a reversible immobilization region. For example, the reversibleimmobilization region can be at least partially delineated or surroundedby a mask region or an annotation mark. In some cases, a first proteinis reversibly immobilized in one region delineated by a first maskregion or annotation mark, and a second protein is reversiblyimmobilized in a second region delineated by a second mark. The processcan be performed, if desired, for 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreproteins, each reversibly immobilized at a specified location.

In some cases, the protein is stored across a large region of thesubstrate surface. For example, the reversibly immobilized protein canbe reversibly immobilized across at least 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, all, or substantially all of at least one surface of thesubstrate, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, all,or substantially all of a reversible immobilization region.

The substrate can be contacted with the protein in the presence of oneor more protein aggregation modifying agents. For example, the substratecan be contacted first with a solution containing one or more proteinaggregation modifying agents, optionally dried, and then contacted withthe protein. Alternatively, or additionally, the protein solution cancontain one or more protein aggregation modifying agents. As yet anotheralternative, one or more protein aggregation modifying agents can alsobe applied after the protein is contacted with the substrate. Thus, theprotein can be contacted with the substrate before a protein aggregationmodifying agent is applied, simultaneously with the application of aprotein aggregation modifying agent, after application of a proteinaggregation modifying agent, or a combination thereof.

The protein can then be reversibly immobilized onto the substrate.Reversible immobilization can occur via a specific binding reaction(e.g., to a ligand present on the substrate), non-specific adsorption,drying of the protein solution and substrate, or via the formation of areversible (e.g., cleavable) covalent bond between the substrate and theprotein. In any case, the protein can be protected from denaturation oraggregation during the reversible immobilization by the presence of oneor more protein aggregation modifying agents.

In some cases, the protein is reversibly immobilized on the substrate bydrying the substrate. In general, drying can be performed by allowing orcausing the volatile solvents (e.g., water, ethanol, etc.) in which theprotein is solubilized to evaporate. Such evaporation can be performedat room temperature and pressure (e.g., about 297 K and 1 atmosphere ofpressure). Alternatively, the evaporation can be performed at highertemperature, lower pressure, or any suitable combination thereof. Forexample, the drying may be performed under lypholization conditions suchas under reduced temperature and pressure. In some cases, the drying maybe performed at a temperature and pressure at which water sublimes fromsolid to gas without transitioning through a liquid phase. Drying cantherefore include air drying, vacuum drying, drying in an oven at anelevated temperature (e.g., 27, 30, 35, 50, 55, 60, 65, 70, 80, or 90°C. or more), and lypholization.

In some cases, e.g., where the reversible immobilization does notinclude a drying step, the substrate containing the reversiblyimmobilized protein can be stored wet. For example, the substrate can beplaced in a container such that the protein is on the substrate and thesubstrate is wet. In some cases, the amount of liquid in the containeris at or below the liquid absorbance capacity of the substrate.

The storage liquid can be the same as the solution in which the proteinwas contacted with the substrate, or can be a different solution. Forexample, the substrate can be contacted with a solution containing aprotein, dried, and then contacted with a storage solution. The storagesolution can be the same as the eluent, or can be a different solution.For example, the substrate can be contacted with a solution containing aprotein, dried, contacted with an amount of eluent at or below theabsorbance capacity of the substrate, optionally stored, and thencontacted with an additional amount of eluent to elute the reversiblyimmobilized protein. In some cases, the storage solution contains one ormore protein aggregation modifying agents.

The substrate containing the reversibly immobilized protein can bestored at any suitable temperature as known in the art for storage ofproteins. For example, the substrate can be stored at room temperature,or at 10, 5, 4, 3, 2, 1, 0, −5, −10, or −20° C., or lower. In somecases, the substrate can be stored at room temperature if it is dry orsubstantially dry and stored at lower than room temperature (e.g., 10,5, 4, 3, 2, 1, 0, −5, −10, or −20° C., or lower) if it is wet.

The protein reversibly immobilized on the substrate can be stored forany suitable length of time. Alternatively, the protein can bereversibly immobilized on the substrate and then used immediately, oralmost immediately (e.g., within 1, 2, 3, 4, 5, 6, 10, 15, 30, 60minutes, or within 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 18, 24, or 48hours). For example, the protein may be contacted with the substrate andthen placed into contact with a second substrate or a membrane totransfer the protein to the second substrate or membrane. As anotherexample, the protein can be contacted with the substrate and then placedinto a transfer apparatus.

Suitable storage times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14,21, 30, 60, or 90 days or more. Suitable storage times further include3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. One of skill in theart will recognize that storage times will vary based on the storageconditions, and the reason for the storage. For example, in some cases,the reversibly immobilized protein may be stored longer at coldertemperatures (e.g., 10, 5, 4, 3, 2, 1, 0, −5, −10, or −20° C., orlower).

As another example, in some cases, the immobilization or storage may beperformed by an entity that sells the protein or provides the protein toothers. Thus, the protein may be stored until an order or request isreceived and the protein has been delivered. In such an example, thestorage time may include the time for transit (e.g., postal service,delivery company, or courier) and receipt of the reversibly immobilizedprotein. Storage time may also include storage by a manufacturer, aconsumer, a distributor, or a retailer.

B. Delivery

Described herein, are methods for delivering a protein from a substrate(e.g., a porous substrate). In some embodiments, the steps of the methodare:

providing a porous substrate containing a reversibly immobilized proteinand a protein aggregation modifying agent; andeluting the reversibly immobilized protein from the porous substratewith an eluent, thereby delivering the protein.

In some cases, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,all, or substantially all of the reversibly immobilized protein iseluted from the porous substrate. In some cases, at least 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, all, or substantially all of the elutedprotein is non denatured or non-aggregated.

Generally, the delivering involves contacting the substrate with aneluent, solubilizing the reversibly immobilized protein, and collectingthe eluent. The contacting can be performed by dipping, coating,pipetting, pouring, spraying, or contacting the substrate with a secondsubstrate, a membrane, or a filter paper that contains eluent. In somecases, the delivery includes delivering the protein into a solution. Insome cases, the delivery includes delivering the protein onto a secondsubstrate, a gel, or a membrane (e.g., a western blot).

In some embodiments, the reversibly immobilized protein is contactedwith an eluent and the eluent, with at least a portion of the protein,is collected in a container. For example, the substrate can be contactedwith an eluent, the protein solubilized, and then the eluent can beseparated from the substrate. For example, the substrate and eluent canbe centrifuged or the eluent can be recovered by filtration, (e.g.,vacuum filtration). Alternatively, the substrate can be contacted withan eluent and subjected to capillary action, vacuum, pressure, or theapplication of an electromagnetic force to cause the eluent, theprotein, or both the eluent and the protein to elute from the substrate.

The electromagnetic force can include an applied voltage or a magneticfield. In some cases, the eluent is at a pH that is above theisoelectric point of the protein and the electromagnetic force isapplied to drive protein toward a positive electrode. In other cases,the eluent is at a pH that is below the isoelectric point of the proteinand the electromagnetic force is applied to drive the protein toward anegative electrode. In some cases, the protein is bound to a chargedmolecule or a plurality of charged molecules and the electromagneticforce is applied to drive the protein toward an electrode of theopposite charge. In some cases, the protein is labeled with a magneticbead or particle, and a magnetic field is utilized to elute the protein.Alternatively, the magnetic bead or particle may be located on a ligandthat has affinity for the protein.

In some embodiments, the reversibly immobilized protein is transferredto a second substrate, a gel (e.g., an acrylamide or agarose gel), or amembrane. For example, the substrate can be contacted with an eluent,and the protein allowed to diffuse onto a second substrate, a gel, or amembrane. Alternatively, the substrate can be contacted with an eluentand subjected to capillary action, vacuum, pressure, or the applicationof an electromagnetic force to cause the eluent, the protein, or boththe eluent and the protein to migrate toward the second substrate, gel,or membrane.

In some cases, the membrane, the gel, or the second substrate includesone or more proteins immobilized thereon. The immobilized protein(s) aregenerally irreversibly immobilized, or substantially irreversiblyimmobilized. For example, in some cases, at least 50%, 60%, 70%, 80%,90%, 95%, 99%, all, or substantially all of the immobilized protein(s)on the second substrate, the gel, or the membrane are not eluted bycontact with the eluents described herein.

In some embodiments, the presence of the transferred protein can then bedetected in or on the second substrate, the gel, or the membrane. Forexample, the presence or absence of a detectable label directly orindirectly linked to the transferred protein can be determined.Alternatively, a binding reagent (e.g., a secondary antibody such as alabeled secondary antibody) can be used to detect the presence orabsence of the transferred protein. In some cases, the transfer of thereversibly immobilized protein from a first substrate to a secondsubstrate, or from the substrate to a gel or a membrane can lead tobinding of the reversibly immobilized protein to a protein that isimmobilized on the second substrate or the membrane, or immobilized inthe gel. In such cases, the detecting of the transferred protein therebydetects the presence of the immobilized protein on the second substrateor the membrane, or in the gel. In some cases, the reversiblyimmobilized protein can be one or more antibodies for detection of oneor more epitopes present on the second substrate or membrane, or presentin the gel. In some cases, such detection methods constitute a westernblot.

V. Articles of Manufacture and Kits

Described herein are articles of manufacture that include one or moreprotein(s), one or more protein aggregation modifying agent(s), and asubstrate. The protein can be any labeled or unlabeled protein asdescribed above. The protein aggregation modifying agent can be anycyclodextrin, non-ionic surfactant, ionic surfactant, zwitterionicsurfactant, non-detergent sulfobetaine, simple sugar, polysaccharide,polyol, organic solvent, aggregation modifying protein, disorderedpeptide sequence, amino acid, oxido-reduction agent, lyoprotectant,cryoprotectant, or chaotropic agent as described above. The substratecan be any of the substrates as described above. In some cases, thesubstrate is a porous substrate.

Also described herein are kits that include one or more (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more) ofthe foregoing substrates, or one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more) of the foregoingarticles of manufacture. In some cases, the kits can further includeinstructions for reversibly immobilizing a protein, instructions for useof one or more components of the kit to detect a protein, orinstructions for eluting a reversibly immobilized protein from acomponent of the kit. In some cases, the kit can include elution buffer,one or more components of an elution buffer, or an elution bufferconcentrate (e.g., 10× elution buffer). In some cases, the kit caninclude one or more protein aggregation modifying agents.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Transfer to a Membrane or a Gel for Detection of ImmobilizedProteins

Using a pipette, 20 μL of antibody (Ab) solution is delivered to one endof a strip of chromatography paper. The antibody solution is prepared in1x Tris-Gly buffer (Bio-Rad) in the presence or absence of 0.25 mMmethyl-B-cyclodextrin (MBCD). This is shown in the top of FIG. 1. Duringwetting of the porous paper, the MBCD protects the Ab from forming Abaggregates. These Ab soaked paper strips are then overlaid on top ofeither polyacrylamide gels (with immobilized antigens) or nitrocellulosemembranes with blotted antigens (shown in FIG. 2). These are thensandwiched between two transfer pads (Bio-Rad Transblot-Turbo pads),soaked in 1xTris Gly buffer. Antibody is then electrophoreticallytransferred from the porous paper substrates to the gel or membranesurface using the Transblot-Turbo. In the case of the gel, the Ab stripsare loaded at a concentration of 1:25, in the case of the nitrocellulosemembrane, the Ab strips are loaded at a concentration of 1:200. Theresult of transfer to a gel is demonstrated in the top of FIG. 3(transfer condition: 0.3 amp for 20 minutes). This image shows thatapproximately 4× as much antibody is delivered from the paper strip thatcontained the MBCD, indicating that in the absence of MBCD, Ab isincreasingly retained in the paper strip, post electrophoresis. This isgraphically demonstrated in the bottom of FIG. 1. The results oftransfer to the membrane are demonstrated in the bottom of FIG. 3(transfer condition: 0.3 amp for 3 minutes). This image shows that inthe absence of Ab, a significant amount of the Ab is delivered from thepaper as an aggregate. This is demonstrated in the “B” lanes of themembranes which show increased “splotchy” background, compared to the“A” lanes which show clean background after transfer indicating atransfer of monomeric Ab. This is graphically demonstrated in the middleof FIG. 1. The two membranes in the bottom of FIG. 3 are blocked witheither 3% BSA (Left), or Pierce Protein Free Blocker (Right).

Example 2 Spatially Controlled Antibody Delivery to a Membrane

A membrane is provided that contains lanes or columns of immobilizedproteins. A porous substrate containing an antibody is contacted withone of the lanes or columns to effect transfer of the antibody to themembrane, as shown in FIG. 4.

Example 3 Spatially Controlled Detection of Target Proteins

A membrane is provided that contains immobilized proteins. A poroussubstrate containing a first antibody localized to a first region, asecond antibody localized to a second region, and a third antibodylocalized to a third region. The first, second, and third regions areseparated by a wax barrier. The substrate is contacted with themembrane, and the substrate:membrane sandwich is placed between acathode buffer pad and an anode buffer pad. A voltage is applied via thecathode and anode to effect transfer of the antibody to the membrane.The binding of the first, second, or third antibody in the regions incontact with the spatially controlled first, second, and third regionsof the substrate leads to spatially controlled detection of targetproteins as shown in FIG. 5.

All patents, patent applications, and other publications, includingGenBank Accession Numbers, cited in this application are incorporated byreference in the entirety for all purposes.

1. A method of delivering a protein, the method comprising: providing aporous substrate comprising a reversibly immobilized protein and aprotein aggregation modifying agent; and eluting the reversiblyimmobilized protein from the porous substrate with an eluent, therebydelivering the protein.
 2. The method of claim 1, wherein the eluentcomprises a buffered solution at a pH above the isoelectric point of thereversibly immobilized protein.
 3. The method of claim 1, wherein theeluent comprises a protein aggregation modifying agent selected from thegroup consisting of a cyclodextrin.
 4. (canceled)
 5. The method of claim1, wherein the eluent comprises tris(hydroxymethyl)aminomethane (Tris),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, methanol,ethanol, or propanol.
 6. The method of claim 5, wherein the eluentcomprises two or more compounds selected from the group consisting oftris-(hydroxymethyl)-aminomethane (Tris),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine, methanol,ethanol, and propanol.
 7. The method of claim 6, wherein the elutingcomprises subjecting the substrate to diffusion, capillary action,vacuum, pressure, or electrophoresis, or contacting the porous substratewith a magnetic particle that has an affinity for the reversiblyimmobilized protein.
 8. The method of claim 7, wherein the methodcomprises eluting at least 50% of the reversibly immobilized proteinfrom the porous substrate with an eluent.
 9. (canceled)
 10. (canceled)11. (canceled)
 12. (canceled)
 13. (canceled)
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. (canceled)
 18. The method of claim 1,wherein 1, 2, 3, 4 or more different proteins are reversibly immobilizedon the porous substrate.
 19. The method of claim 1, wherein the poroussubstrate comprises a composition selected from the group consisting ofwhatman paper, paper, a cellulose filter, a glass microfiber filter,nitrocellulose, polyvinylidene difluoride, a sintered glass, a sinteredpolymer, a sintered metal, a spunbound polyester, rayon, nylon, a porouspolymer monolith, a porous polymer bead, a capillary wicking bed,natural or synthetic sponge, and fiberglass.
 20. (canceled) 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)26. (canceled)
 27. (canceled)
 28. (canceled)
 29. The method of claim 1,wherein the protein is reversibly immobilized on the porous substratefor at least about 1 week, 1 month, or 6 months.
 30. (canceled) 31.(canceled)
 32. (canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)36. The method of claim 1, wherein the protein aggregation modifyingagent is selected from the group consisting of a cyclodextrin.
 37. Themethod of claim 36, wherein the: cyclodextrin is selected from the groupconsisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, andsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin.
 38. A method of storing aprotein the method comprising: providing a porous substrate and aprotein aggregation modifying agent; contacting the porous substrate inthe presence of the protein aggregation modifying agent with a solutioncontaining a protein; and reversibly immobilizing the protein on theporous substrate, thereby storing the reversibly immobilized protein onthe porous substrate.
 39. The method of claim 38, wherein at least 50%of the reversibly immobilized protein is substantially non-denatured andsubstantially non-aggregated.
 40. (canceled)
 41. The method of claim 38,wherein the porous substrate comprises a composition selected from thegroup consisting of whatman paper, paper, a cellulose filter, a glassmicrofiber filter, nitrocellulose, polyvinylidene difluoride, a sinteredglass, a sintered polymer, a sintered metal, a spunbound polyester,rayon, nylon, a porous polymer monolith, a porous polymer bead, acapillary wicking bed, natural or synthetic sponge, and fiberglass. 42.(canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)
 46. (canceled)47. (canceled)
 48. (canceled)
 49. The method of claim 38, wherein thereversible immobilization comprises drying the porous substrate aftercontacting the porous substrate in the presence of the proteinaggregation modifying agent with the solution containing the protein.50. (canceled)
 51. (canceled)
 52. (canceled)
 53. (canceled) 54.(canceled)
 55. (canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled)59. (canceled)
 60. (canceled)
 61. (canceled)
 62. (canceled) 63.(canceled)
 64. The method of claim 38, wherein the protein aggregationmodifying agent is a cyclodextrin selected from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin,(2,3,6-tri-O-methyl)-β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin,(2-hydroxy)propyl-γ-cyclodextrin, random methyl-β-cyclodextrin, randommethyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl-γ-cyclodextrin, 6-monodeoxy-6-monoamino-β-cyclodextrin,sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetylβ-cyclodextrin, succinyl α-cyclodextrin, succinyl β-cyclodextrin,succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin,succinyl-(2-hydroxypropyl)-β-cyclodextrin, andsuccinyl-(2-hydroxypropyl)-γ-cyclodextrin.
 65. An article of manufacturecomprising a porous substrate, a protein reversibly immobilized on asurface of the substrate, and a protein aggregation modifier, whereinthe article is made by the method of claim
 38. 66. The article of claim65, wherein the protein reversibly immobilized on the surface of thesubstrate is dry.
 67. The article of claim 65, wherein the substrate isat least about 5-15 cm wide and at least about 5-15 cm long 68.(canceled)
 69. (canceled)
 70. (canceled)
 71. (canceled)
 72. (canceled)